[Molecular detection assays for 2012 identified novel human coronavirus (HCoV) and probe modification with locked nucleic acid (LNA)].

Zhou, Wei-Min; Lu, Rou-Jian; Gong, He-Yuan; Li, Hui; Duan, Xi-Jie; Yang, Yang; Tan, Wen-Jie

OBJECTIVE To develop and optimize the molecular detection assays for recently identified human coronavirus (HCoV) infection. METHODS Based on the 208 base pair(bp) sequence of novel HCoV reported by HPA of UK, we designed and obtained several pairs of primer (F-1, R-1; F-2, R-2) and Taqman probes (TZ1,TZ2) for detection of novel HCoV. Two of probes were modified with LNA (LNA-TZ1, LNA-TZ2). Then, RT-PCR and various real time RT-PCR assays were developed and optimized in this study. We also compared our assays with the real time RT-PCR assays reported recently by Europe team based on upE or ORF1b target. RESULTS The RT-PCR or real time RT-PCR assays for novel HCoV were developed without cross-reactivity with other HCoV and several common respiratory viruses using clinical specimen panel. The analytical sensitivity of assays were less than 50-500 copies per reaction and the detection was improved when Taqman probe modified with LNA-tagged, compared to no LNA-tagged in real time RT-PCR assays. The upE and LNA-TZ1 based assays were better than others. CONCLUSION The molecular detection sensitivity and specificity of TaqMan-based real time PCR assay could be improved when probe tagged with LNA. The upE or LNA-TZ1 based real time RT-PCR assay was recommend for detection of novel HCoV. This study laid a foundation for improving the performance of novel HCoV detection.